The goal of this research project is to determine the change in protein structure related to removal of bound water. All protein structures presently known at or near atomic resolution are highly hydrated. This application proposes to study a protein at various levels of reduced hydration: to step-by-step strip away water molecules to understand the details of their effect on protein structure. Efforts to predict protein structure through computational chemistry often resort to modeling the protein as if it were in vacuum and not surrounded by water. This project will provide important and unique experimental data to compare with such calculations. Dehydration of proteins occurs during freeze-drying of protein pharmaceuticals now produced through biotechnology. Rehydration and recovery of activity is a problem that will be better understood when the structural effects of protein dehydration are understood. A growing area of biotechnology involves the use of enzymes in organic solvents. Efforts have begun to design enzymes for these unusual environments. To aid these design efforts, this study will also describe the details of the structure of an enzyme in organic solvents. X-ray crystallographic methods will be used to determine the crystal structure of the enzyme bovine pancreatic ribonuclease A when dried in air, and when dehydrated in several different organic solvents.